The present invention relates to the title aspects of the major grass pollen allergen Phl p I and IgE-binding epitopes present in this allergen and corresponding haptens. The invention also relates to fragments, including IgE-binding haptens, from other grass and monocotyledonic plant allergens containing the IgE binding epitopes of Phl p I. The invention is primarily concerned with epitopes that normally are found in one or more group I allergens.
Up to 20% of the population in industrialized countries suffer from Type I allergic symptoms (rhinitis, conjunctivitis, asthma bronchiale) (Myamoto et al., 1992). The crosslinking of IgE which is bound to mast cells and basophils via the high affinity receptor Fcxcex5RI is the key event leading to release of biological mediators such as histamine (Segal et al., 1977). The crosslinking event by allergens represents, therefore, a potential target for therapy of Type I allergy. Such therapeutical approaches could either use portions of the IgE-molecule or other ligands, to interfere with the binding of IgE to the high affinity Fcxcex5-receptor, or reagents to block the subsequent signal transduction cascade thus preventing the degranulation of mast cells and basophils (Dreskin et al., 1988). An additional possibility for specific therapy would be to use haptens derived from complete allergens which by binding to IgE monovalently could block the crosslinking of IgE (Valenta et al., 1993a). IgE-haptens could also be used to modulate the immune response or to induce tolerance by immunotherapy with a minimum of anaphylactic side effects. Haptens can be obtained from complete allergens by proteolytic digestion. However, this often results in a mixture of fragments and enzymes that are difficult to characterize. Synthesis of peptides based on the amino acid sequence of the allergens, is an alternative approach. Recently a number of cDNAs coding for important allergens (Scheiner et al., 1992) were isolated which can be used to determine IgE-epitopes by molecular biological techniques.
Grass pollen allergy is spread world wide and according to the prevalence of grass pollen allergy it can be expected that 75% of all allergic patients suffer from grass pollen allergy (Freidhoff et al., 1986).
Among the grass pollen allergic patients more than 90% display IgE-reactivity with group I allergens (Freidhoff et al., 1986; Valenta et al., 1992).
The full amino acid sequences and nucleotide sequences of the major grass pollen allergens have been known for some time (timothy grass Phl p I (Laffer et al., 1993), rye grass (Lolium perenne) Lol p I (Perez et al., 1990; Griffith et al., 1991; University of Melbourne WO-A-9203550,; Brunet C et al., International Symposium on Molecular Biology of Allergens and the Atopic Response, Quebec City, Canada, Feb. 18-22, 1995; Lamontagne P et al., International Symposium on Molecular Biology of Allergens and the Atopic Response, Quebec City, Canada, Feb. 18-22, 1995), and from rye from timothy grass (Phleum pratense) Sec c I (Laffer et al., unpublished data).
During the priority year the determination of clones 80, 97 and 98 as carriers for a group I conserved IgE binding epitope has been described (Ball et al., 1994a, b, c; Laffer et al., 1994;).
Definition
The term IgE-hapten identifies short allergen fragments on which only one IgE-antibody with a given specificity is allowed to bind. A real IgE-binding hapten will give no histamine release because it contains the binding site for exclusively one IgE antibody. The term epitope in the context of the present invention refers to an IgE-epitope if not otherwise specified. An epitope may be located on either an IgE-hapten or a longer polypeptide comprising several IgE-binding sites/epitopes. The term IgE preferentially refers to human IgE.
The objectives of the invention are to provide simple, better and more reliable in vitro an in vivo tests for grass pollen allergy as well as improved therapeutic methods for this disease.
The Invention
A first aspect of the invention is a recombinant DNA molecule comprising a nucleotide sequence (I) which codes for a polypeptide displaying the antigenicity of at least one of the Phi p I epitope clones 28 (SEQ ID NO: 26), 34 (SEQ ID NO: 15), 41 (SEQ ID NO: 24), 42 (SEQ ID NO: 27), 43 (SEQ ID NO: 14), 45 (SEQ ID NO: 13), 50 (SEQ ID NO: 18), 52 (SEQ ID NO: 28), 64 (SEQ ID NO: 20), 80 (SEQ ID NO: 5), 85 (SEQ ID NO: 22), 86 (SEQ ID NO: 23), 95 (SEQ ID NO: 17), 97 (SEQ ID NO: 7), 98 (SEQ ID NO: 9), 103 (SEQ ID NO: 19), 108 (SEQ ID NO: 25), 109 (SEQ ID NO: 21), 113 (SEQ ID NO: 12), and 114 (SEQ ID NO: 16) with the amino acid sequences defined in SEQ ID NOS: 5, 7, 9 and 12-28 and preferably being derived from grasses or monocotyledonic plants, or a nucleotide sequence (TI) which hybridizes with a nucleotide sequence (I) under conditions of high stringency. The recombinant DNA molecule comprises also degenerate variants of these nucleotide sequences.
The recombinant DNA molecule may also contain a nucleotide sequence which codes for a polypeptide having antigenic crossreactivity and a high degree of homology, preferable  greater than 50% such as  greater than 60% or  greater than 75%, with Phi p I epitopes from grasses or other monocotyledonic plants, preferably those defined by the amino acid sequences given in SEQ ID NOS: 5, 7 and 9-28.
A second aspect of the invention is a recombinant DNA expression vector or cloning system comprising an expression control sequence operatively linked to any of the recombinant molecules defined above.
A third aspect of the invention is a host cell containing a recombinant molecule or vector according to the first or second aspect, respectively.
A fourth aspect of the invention is a recombinant or synthetic protein or polypeptide displaying the antigenicity of a Phi p I epitope, in particular comprising as an essential part a Phi p I epitope of at least one of the sequences set out in SEQ ID NOS: 5, 7 and 9-28. The protein or polypeptide may be fused to an additional polypeptide, such as xcex2-galactosidase, GST or lambda cII protein or any other polypeptide that can be expressed as a fusion protein in prokaryotic or eukaryotic cells.
In the inventive poly/oligonucleotides and proteins/polypeptides, at least one of the sequences defined in SEQ ID NOS: 5, 7 and 9-28 constitutes an essential part. For the poly/oligonucleotides this means that each of them should not be longer than half of the DNA sequence coding for the full length Phi p I allergen (SEQ ID NO: 10) and preferably containing a nucleotide sequence coding for at least one Phi p I epitope, such as being present in the Phi p I fragments specified in SEQ ID NOS: 5, 7 and 9-28. The inventive oligo/polynucleotides changes are often shorter than 25% of the DNA encoding for the full length phi p I allergen.
For the inventive proteins and polypeptides xe2x80x9cessential partxe2x80x9d means that each of them should not be longer than half of the full length Phi p I allergen and preferably also contain at least one Phi p I epitope, such as one or more of the epitopes defined by the fragments of the full length Phi p I allergen specified in SEQ ID NOS: 5, 7, 9 and 12-28. The inventive proteins and polypeptides are often shorter than 25% of the full length Phi p I allergen.
By the expression xe2x80x9ca polypeptide displaying the antigenicity of at least one of the clones 28, 34, 41, 42, 43, 50, 52, 64, 80, 85, 86, 95, 97, 98, 103, 108, 109, 113, 114xe2x80x9d is meant any peptide portion displaying at least one epitope defined by these clones and being recognizable immunologically. It can be envisaged that polypeptides exhibiting Phl p I epitopes may be derivatized to carry analytically detectable groups or water-soluble or water-insoluble solid phases suitable for immunoassays of antibodies directed against them, e.g. IgA, IgD, IgE, IgM or IgG antibodies. In aspects of the invention relating to in vitro diagnostics (see below) the inventive peptides may be a) linked to a water-insoluble phase by physical adsorption or a covalent bond, or b) conjugated covalently to an analytically detectable group (label).
The fifth aspect the invention is an in vitro method for diagnosing allergy to plant proteins by determining humoral antibodies directed towards the plant proteins. The allergies concerned are mostly against grass pollen. The relevant antibodies are mostly of the IgE class but IgG antibodies may also give information about the allergy. In general this method comprises contacting a body fluid sample derived from a patient with an inventive polypeptide. The amounts and conditions are selected so that an immune complex between the polypeptide and antibodies in the sample are formed in an amount that is a function of the amount of antibodies in the sample. The immune complex is then measured in a per se known manner.
More specifically a preferred method of the fifth aspect comprises contacting a body fluid sample containing the Igs concerned, e.g. IgG or IgE, with a polypeptide according to the invention and an anti-IgE antibody so as to form the immune complex containing peptide:IgE:anti-IgE. Normally either the peptide or the anti-IgE is linked to a solid phase that is insoluble or insolubilizable in the assay media so that the complex can be separated from the assay media. The determination step in these variants may be performed by use of an analytically detectable group (label) that either is covalently linked to the anti-IgE antibody (in case the peptide is linked to the solid phase) or to the peptide (in case the anti-IgE antibody is linked to the solid phase). In case IgG antibodies are to be determined anti-IgG replaces anti-IgE.
A sixth aspect of the invention is a method employing measuring, preferably in vitro, the cellular reaction against a Phl p I epitope. The method comprises using a recombinant or synthetic polypeptide as defined for the fourth aspect to stimulate the cellular reaction. Cellular reactions to be measured are histamine release and T cell proliferation (by 3H thymidine uptake).
The samples used in the above-mentioned methods are often derived from blood such as whole blood, serum and plasma, although also other body fluids containing Igs may be used (tears etc).
Commonly accepted solid phase forms useful for immunoassays are walls of microtitre wells, spheres, rods, sheets, strips, pads etc. The solid phase may be porous or non-porous. The material in the solid phase may be a polymer selected among polysaccharides and their derivatives, for instance dextran, pullulan, agarose, cellulose etc, or synthetic polymers, preferably vinyl polymers, such as polyacrylamides, polyacrylates, polystyrene, polyvinyl alcohol etc. The polymers in question are often cross-linked, particularly in case the base polymer as such is water-soluble. Examples of analytically detectable groups are isotopes, enzymes, enzyme substrates, fluorophors, haptens, biotin etc.
A seventh aspect of the invention is a method for the treatment of a mammal, such as a human, which has a pollen allergy by administering a therapeutically effective amount of a recombinant or synthetic polypeptide as defined above. Illustrative examples of the therapeutic aspect of the invention are: a) passive therapy of effector organ (nose, conjunctiva, and lung) to prevent mediator release upon subsequent exposition to the complete allergen, and b) use of the peptides as safe tools for active immunotherapy because a single IgE epitope as such do not release mediators so that high doses can be applied. See further in the Discussion part below. The administration routes will be as commonly applied for current hyposensitization. The doses are likely to be in the xcexcg/ml-range per kg body weight, i.e. 10-100 xcexcg per kg body weight.